Vitrification is the process of direct transition from a liquid to an amorphous glassy state and is often utilized to preserve biological materials by cooling them to cryogenic temperatures at high cooling rates. At cryogenic temperatures, vitrification technique avoids the damaging effects of ice crystals, which are known to form during conventional cryopreservation using slow cooling rates. However, in order to avoid ice-nucleation during cooling, extremely high and potentially toxic concentrations (6-8M) of cryoprotectants (CPAs) are required. Among the most commonly used CPAs are dimethyl sulfoxide (DMSO), glycerol, ethylene glycol (EG) and 1,2-propanediol (PROH). As a result, multiple steps and complex elaborate protocols are required to load and unload CPAs into cells. Therefore, alternative approaches to achieve vitrification without the need to expose biologics to high concentrations of CPA at non-cryogenic temperature have been sought over the years.
It is known that in order to achieve cryogenic vitrification at lower CPA concentrations, ultra-fast heat transfer rates are required. Heat transfer rates can be increased by reducing the sample volume and/or by increasing the cooling rate. A number of techniques have been utilized to increase the cooling rate such as employing thin straws or ultra-thin films to minimize the volume to be vitrified. Patent application US 2013/0157250 A1, published Jun. 20, 2013 discloses a method for cryogenic vitrification of human spermatozoa in low CPA concentration employing a thin straw. More recently, taking advantage of the high thermal conductivity of quartz crystal (QC) capillaries, patent application US 2013/0260452 A, published Oct. 3, 2013 in which N. Chakraborty is a common inventor, discloses a method for vitrification of mammalian cells in low CPA concentration medium at ultra-rapid cooling rates.
Anhydrous vitrification at ambient temperatures may also be an alternative strategy for preserving biological materials. In nature, a wide variety of organisms can survive extreme dehydration which correlates in many cases with the accumulation of large amounts (as much as 20% of their dry weight) of glass forming sugars such as trehalose and sucrose in intracellular space. Such “glass forming” sugars need to be present on both sides of the plasma membrane to provide protection against the damaging effects of desiccation. Desiccation techniques dramatically limit or arrests the material's biochemical processes in a glassy matrix. Despite the success in vitrifying many biological materials such as proteins by anhydrous vitrification, broader applications to cellular materials still requires one to increase the desiccation tolerance of the cells.
Methods to enhance desiccation tolerance include utilizing improved vitrification medium containing trehalose, glycerol and sucrose. While improved methods for loading cells with protective agents are helpful, there is a further need to develop techniques to minimize cellular injury during desiccation. Injury and degradation may result from the high sensitivity of cells in general to prolonged exposure to osmotic stress during dry processing. Osmotic stress can cause cell death at relatively high moisture content even in the presence of protective sugars like trehalose.
The most common approach to desiccating cells involves drying in sessile droplets with suspended cells. However, desiccation using evaporative drying of sessile droplets is inherently slow and non-uniform in nature. A glassy skin forms at the liquid/vapor interface of the sample when the cells are desiccated in glass forming solutions. This glassy skin slows and ultimately prevents further desiccation of the sample beyond a certain level of dryness and induces significant spatial non-uniformity of the water content across the sample. As a result, cells trapped in the partially desiccated sample underneath the glassy skin may not vitrify but degrade due to high molecular mobility.
U.S. Pat. No. 7,883,664, in which N. Chakraborty is a common inventor, discloses a method for enhancing desiccation rate by employing microwave drying. Further, U.S. Pat. No. 8,349,252, in which N. Chakraborty is a common inventor, discloses a vitrified composition comprising trehalose for vitrification by microwave drying. However, the method cannot achieve continuous drying as the biological material's temperature increases continuously to unsafe levels and requires a complicated process control. Chakraborty et al. have also employed spin drying technique to create ultra-thin films and successfully vitrified hamster ovary cells in trehalose medium. However, this approach still suffers from the limitations that the desiccation cannot be uniformly performed across the entire sample surface. Further, the film has to be ultra-thin to vitrify successfully.
The development of a fast and practical desiccation technique to achieve very low and uniform final moisture levels across the sample might overcome the shortcomings of the anhydrous vitrification techniques. Dry preservation suffers from a major limitation in long-term storage due to the degradation of the biological material by cumulative chemical stresses encountered as the vitrification solution gets concentrated in the extra-cellular space. This results in irreversible cell damage before the cells and the vitrification solution can reach a suitably low moisture content to become glassy. Therefore, there exists a need for improved vitrification medium to vitrify biological materials by fast drying while maintaining the material's viability. A fast desiccation method with improved cell viability will tremendously facilitate long term storage of biological materials at non-cryogenic temperatures as well as overcome the challenges associated with cryogenic vitrification and storage technologies.